As a technique for expressing a recombinant protein in a microbial host, an expression system using Escherichia coli (E. coli) as a host has been generally and widely used. This is because E. coli is extremely easy to handle in a laboratory. More specifically, E. coli is confirmed as a safe microbial host and proliferates at a high rate, and its molecular biological operations in a laboratory are well established. On the other hand, development of host microorganisms having usefulness and advantages over E. coli in view of recombinant protein expression has progressed.
Microorganisms of the genus Rhodococcus are not pathogenic, except a few, and easily cultured in an ordinary laboratory. In addition to such essential conditions, they have the function as microbial catalysts, which is considered to be extremely useful from an industrial point of view. For these reasons, recently, various molecular biological techniques have been developed by use of such microorganisms. For example, in an attempt to add a further useful function to the microorganisms, techniques involving gene recombination has been developed. As a result, a shuttle vector was established which can replicate autonomously both in E. coli and in a microorganism of the genus Rhodococcus (R, De Mot et al., Microbiology 143, 3137-3147, (1997)). Furthermore, there is a report that a transposable transposon is present in a microorganism of the genus Rhodococcus (I, Nagy et al., J. Bacteriol. 179, 4635-4638 (1997)). Thus, it is expected to improve the microorganism in function, for example, by destroying the gene or integrating an exogenous gene into the chromosome.
In an attempt to further improve a microbial catalytic action based on such a molecular biological establishment, development of a vector for expressing a recombinant protein has been underway (JP Patent Publication (Kokai) No. 10-248578 A (1998)).
A microorganism of the genus Rhodococcus, namely, Rhodococcus erythropolis is not only useful as a microbial catalyst but also advantageous in that it can grow under a low temperature condition of 4° C. For this reason, it is expected that Rhodococcus erythropolis may produce a recombinant protein or the like in a temperature range where E. coli could not be used. Development of an inducible expression vector has been underway for such a purpose (the application already filed by Tamura, on Aug. 12, (2002)).
However, the cell wall of a microorganism of the genus Rhodococcus is particular and rigid in structure compared to those of other gram-positive bacteria. Therefore, extraction of a cellular content from the microorganism is complicated and difficult compared to the case of E. coli. More specifically, a microorganism of the genus Rhodococcus has an extremely strong resistance to a cell-wall lytic enzyme used generally for microbial cell lysis, such as lysozyme. Examples of a cell lysis method include a method of exposing cell wall to a high-concentration antibiotic, such as penicillin, for a predetermined time to weaken the cell wall and then being subjected to cell lysis with lysozyme, and a method of applying ultrasonic treatment to bacterial cells for a long time to physically destroy them. However, these methods are complicated in process, it is difficult to treat a large amount of cells, and specimens are not likely to be treated uniformly. These problems are significant in view of industrial use. The effectiveness of an antibiotic such as penicillin is brought by inhibiting a de-novo synthesis of a cell wall and therefore the cell wall completed in synthesis is not affected by such an antibiotic. Therefore, the effect of such an antibiotic is considered to be low in low-temperature conditions where rapid growth is not expected.
It is known that the cell wall structure of a microorganism of the genus Rhodococcus is commonly seen in bacteria of the genus Corynebacterium (C. E. Barry III et al., Prog. Lipid Res. 37, 143-179 (1988)) and an invention similar to the present invention has been made in view of an object of facilitating a molecular biological operation such as transformation (JP Patent Publication (Kokoku) No. 01-003475 B (1989), T. Hirasawa et al., J. Bacteriol. 182, 2696-2701 (2000)).